The goal of this research proposal is to study in detail the recently established T-cell costimulatory roles of the adhesion pathways LFA-l/ICAM-1, VLA-4/VCAM-1 and CD2/LFA-3 + CD59. The main objective of this research proposal is to determine the role of these costimulatory adhesion pathways in the generation of functionally distinct human T helper subsets. To reach the stated goal, there are two specific aims: 1) Analysis of the role of costimulatory adhesion pathways LFA-l/ICAM-1, VLA-4/VCAM-1 and CD2/LFA-3 + CD59, in post-thymus differentiation and maturation of human CD4+ T-cells under in vitro cell culture conditions. In particular focussed on regulation of cell surface expression of activation and differentiation antigens, and production of T-cell cytokines. 2) Analysis of early signal transduction pathways involved in the costimulatory function of the costimulatory adhesion pathways LFA-l/ICAM-1, VLA-4/VCAM-1, and CD2/LFA-3 + CD59. Special emphasis will be given of the regulation of phosphotidylinositol hydrolysis and the role of tyrosine protein kinases and their phosphorylated substrates. The unique characteristic of this study will be the utilization of affinity purified natural ligands, namely ICAM-1, VCAM-1, LFA-3 and CD59, to study the contribution of the various pathways in isolation. Another distinctive feature of this study will be that it will be restricted to resting human CD4+ T-cells isolated from peripheral blood from healthy volunteers. Major emphasis will be given to the CD45RA+CD45RO- (putative phenotypic markers for "naive" T-cells) and CD45RA-CD45RO+ (putative phenotypic markers for "memory" T-cells) subsets of CD4+ T-cells, which will be isolated through negative magnetic selection with monoclonal antibodies (mAbs) and magnetic beads. The investigations outlined in this research proposal will contribute to a more detailed insight in the mechanisms involved in T-cell differentiation/maturation. This knowledge might ultimately be used to generate specific T-cell effector subpopulations, which may be of value either therapeutically in specific disease situations or prophylactically in vaccination protocols.